Angiogenesis induced by tumor necrosis factor-agr; is mediated by α4 integrins

Tumor necrosis factor-α (TNF-α) and fibroblast growth factor-2 (FGF-2 or bFGF) are potent stimulators of angiogenesis. TNF-α, but not FGF-2, can induce the expression of vascular cell adhesion molecule-1 (VCAM-1) on the surface of endothelial cells. The soluble form of VCAM-1 has recently been demonstrated to function as an angiogenic mediator. Here we demonstrate that monoclonal antibodies directed against VCAM-1 or its α4 integrin counter-receptor inhibited TNF-α-induced endothelial cell migration in vitro. Angiogenesis induced in vivo in rat corneas by TNF-α was inhibited by a neutralizing antibody directed against the rat α4 integrin subunit. A peptide antagonist of the a4 integrins blocked TNF-α-induced endothelial cell migration in vitro and angiogenesis in rat corneas in vivo. No inhibition by the antibodies or peptide antagonist was observed either in vitro or in vivo when FGF-2 was used as the stimulus. The peptide antagonist did not inhibit TNF-a binding to its receptor nor did it block the function of αvβ3, an integrin previously implicated in TNF-a and FGF-2 mediated angiogenesis. These results demonstrate that angiogenic processes induced by TNF-α are mediated in part by agr;4 integrins possibly by a mechanism involving the induction of soluble VCAM-1.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41761/1/10456_2004_Article_188219.pd

Mitigating Permeability and Absorption Risks in Drug Discovery

Adequate permeability is essential for good oral absorption and proper interpretation of pharmacokinetic and pharmacological data. Permeability-limiting absorption, however, is a complex phenomenon involving multiple mechanisms and organs ranging from gastro-intestine, liver, kidney to brain blood barrier, but the options for pharmaceutical improvement of permeability are quite limited. In this article, the comprehensive in silico, in vitro and in vivo/ex vivo/in situ tools to assess permeability were reviewed, alongside their advantages and limitations. The integrated strategy to mitigate the permeability-related absorption or safety risks at the different stages of drug discovery and development processes was presented. Several key points relating to the in vivo predictive impact of in vitro permeability tools and the caveats when dealing with challenging discovery compounds were also addressed, using drug discovery cases and statistics in Novartis

Towards prediction of in vivo exposure from a permeability perspective using a 96-well Caco-2 assay

In this study we systematically validated a reproducible 96-well Caco-2 assay via an extended test set of 93 marketed drugs consisting of diverse transport mechanisms and quantified by LC/MS/MS. We sought to investigate the predictive utility of the widely-accepted Caco-2 permeability assay while dealing with the impact of solubility and recovery on challenging discovery compounds. We observed that paracellular compounds could be flagged by absorptive permeability (logPapp(A-B) <-5.5 cm/s) and physicochemical property space (clogP <1). An examination of 8000 Novartis discovery compounds revealed 14% were rejected for testing based on low aqueous solubility, while 13% were subject to low recovery (<30%). Compound loss in the assay was examined using a comparison between cell monolayer and artificial membrane (PAMPA), while bovine serum albumin (0.5% BSA in both donor and acceptor compartments) was utilized to improve recovery in the assay. The caveat to using Vitamin E TPGS to reduce solubility drop-offs was evaluated. The second focus of this study was to investigate the advantages and limitations of the current 96-well Caco-2 screening assay for predicting in vivo exposure from the permeability perspective in the drug discovery stage. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with sub-optimal physicochemical properties, Caco-2 data successfully projected low exposure. This platform set the stage for mechanistically evaluating subsets of compounds towards improving in vitro-in vivo correlations

Steroid Receptor Coactivator-1 from Brain Physically Interacts Differentially with Steroid Receptor Subtypes

In vitro studies reveal that nuclear receptor coactivators enhance the transcriptional activity of steroid receptors, including estrogen (ER) and progestin receptors (PR), through ligand-dependent interactions. Whereas work from our laboratory and others shows that steroid receptor coactivator-1 (SRC-1) is essential for efficient ER and PR action in brain, very little is known about receptor-coactivator interactions in brain. In the present studies, pull-down assays were used to test the hypotheses that SRC-1 from hypothalamic and hippocampal tissue physically associate with recombinant PR or ER in a ligand-dependent manner. SRC-1, from hypothalamus or hippocampus, interacted with PR-A and PR-B in the presence of an agonist, but not in the absence of ligand or in the presence of a selective PR modulator, RU486. Interestingly, SRC-1 from brain associated more with PR-B, the stronger transcriptional activator, than with PR-A. In addition, SRC-1 from brain, which was confirmed by mass spectrometry, interacted with ERα and ERβ in the presence of agonist but not when unliganded or in the presence of the selective ER modulator, tamoxifen. Furthermore, SRC-1 from hypothalamus, but not hippocampus, interacted more with ERα than ERβ, suggesting distinct expression patterns of other cofactors in these brain regions. These findings suggest that interactions of SRC-1 from brain with PR and ER are dependent on ligand, receptor subtype, and brain region to manifest the pleiotropic functional consequences that underlie steroid-regulated behaviors. The present findings reveal distinct contrasts with previous cell culture studies and emphasize the importance of studying receptor-coactivator interactions using biologically relevant tissue

The macrophage: the intersection between HIV infection and atherosclerosis

HIV-infected individuals are at increased risk of coronary artery disease (CAD) with underlying mechanisms including chronic immune activation and inflammation secondary to HIV-induced microbial translocation and low-grade endotoxemia; direct effects of HIV and viral proteins on macrophage cholesterol metabolism; and dyslipidemia related to HIV infection and specific antiretroviral therapies. Monocytes are the precursors of the lipid-laden foam cells within the atherosclerotic plaque and produce high levels of proinflammatory cytokines such as IL-6. The minor CD14+/CD16+ “proinflammatory” monocyte subpopulation is preferentially susceptible to HIV infection and may play a critical role in the pathogenesis of HIV-related CAD. In this review, the central role of monocytes/macrophages in HIV-related CAD and the importance of inflammation and cholesterol metabolism are discussed